Evidence for allosteric regulation of succinyl-CoA synthetase

Abstract

We have previously reported that distinctly different concentrations of GDP stimulate the phosphorylation and dephosphorylation of p36, the alpha-subunit of succinyl-CoA synthetase (SCS) in Dictyostelium discoideum. In this present study, we have investigated the mechanism underlying these dual effects of GDP. Dephosphorylation of p36 is induced by relatively high levels of GDP and is coincident with the formation of GTP. This indicates that, at high concentrations, GDP serves as a substrate of SCS. However, 100-fold lower concentrations of GDP, which do not bind to the catalytic site to induce SCS dephosphorylation, stimulate p36 phosphorylation. This stimulation is not diminished by dilution of the sample, and is retained during purification of the protein. Gel-filtration analyses indicate that SCS in our system behaves as a non-interacting alpha beta dimer, the hydrodynamic behaviour of which is not altered by the presence of added GDP. The data indicate that altered protein-protein interactions do not account for the stimulation of p36 phosphorylation by low GDP concentrations. We propose that GDP functions as an allosteric regulator of SCS, and experiments using guanosine 5'-[beta-thio]diphosphate (GDP[S]) are shown to distinguish further the allosteric and catalytic binding sites.

Succinyl Coenzyme A Synthetase of Pseudomonas aeruginosa with a Broad Specificity for Nucleoside Triphosphate (NTP) Synthesis Modulates Specificity for NTP Synthesis by the 12-Kilodalton Form of Nucleoside Diphosphate Kinase

“…In contrast, Scs from eukaryotes such as pig heart (8) or Dictyostelium discoidium (1,25,26) is known to generate GTP. However, both ATP-and GTP-specific Scs isoforms have been characterized from multicellular eukaryotes (8) and, in general, the nucleotide preference varies widely within organisms (11,20).…”

“…The Scs ␣ subunit can be efficiently autophosphorylated either in presence of ATP or in presence of GTP, and small quantities of GDP or ADP can enhance such phosphorylation, while larger concentrations inhibit it, suggesting that intracellular concentrations of ADP and GDP modulate the interconversion of ATP and GTP by Scs. The stimulatory effect of low concentrations of GDP and ADP in the autophosphorylation of the ␣ subunit of Scs may be due to binding to a high-affinity allosteric site (3,26) of Scs. The stimulation of Scs phosphorylation by GDP is well documented in the case of eukaryotes (8,25), but it also occurs in P. aeruginosa.…”

Novel mechanisms of Escherichia coli succinyl-coenzyme A synthetase regulation

“…Partial purification of SCS on hydroxyapatite was performed as previously described (2). The stability of the radiolabel in the reaction mixture was monitored by thin-layer chromatography on polyethyleneimine-cellulose MN 300 plates in 0.75 M KH 2 PO 4 , pH 3.4, as previously described (19).…”

“…Phosphorylation assays were performed at room temperature in buffer A and have been previously described in detail (18)(19)(20). Briefly, phosphorylation was initiated by incubating samples with either [␥-…”

“…The reversible nature of its reactions and the apparent absence of any higher-order regulation would suggest that SCS is not a critical control point for the flow of substrates through the cycle. However, recent experiments by Um and Klein (18)(19)(20) have provided evidence that the activity of Gform enzymes is allosterically regulated. At concentrations above 5 ϫ 10 Ϫ6 M, GDP binds to the catalytic site of the enzyme.…”